A Genetically Encoded Assay System to Quantify O-GlcNAc Transferase (OGT) Activity in Live Cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• O-GlcNAc transferase (OGT) is crucial for cellular functions, regulating nucleocytoplasmic proteins.
• OGT dysregulation is linked to diseases like cancer and neurodegeneration.
• Existing methods lack direct cellular OGT activity assays.

Purpose of the Study:

• To develop a genetically encoded reporter for real-time cellular OGT activity measurement.
• To enable the discovery and characterization of OGT activity modulators.
• To investigate the impact of metabolites on OGT activity.

Main Methods:

• Engineered a reporter system based on OGT's cleavage of Host Cell Factor-1 (HCF-1).
• Utilized fluorescent proteins linked to nuclear localization/export signals to report cleavage.
• Quantified OGT activity by measuring fluorescent protein distribution shifts.
• Validated the reporter with known OGT pathway modulators and metabolites.

Main Results:

• Demonstrated a novel OGT cellular activity reporter (OGT-CAR) system.
• Showcased the reporter's sensitivity and precision in detecting OGT activity changes.
• Assessed dose- and time-dependent effects of OGT modulators and metabolites.
• Successfully measured OGT activity within living cells.

Conclusions:

• The OGT-CAR system provides a direct, genetically encoded measure of cellular OGT activity.
• This reporter facilitates the identification of novel therapeutic targets and drugs.
• The OGT-CAR system is a valuable tool for studying O-GlcNAc pathway regulation in health and disease.